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Conserved amino acids in each subunit of the heteroligomeric tRNA m1A58 Mtase from Saccharomyces cerevisiae contribute to tRNA binding.

Ozanick SG, Bujnicki JM, Sem DS, Anderson JT - Nucleic Acids Res. (2007)

Bottom Line: Yeast strains expressing trm6 and trm61 mutants exhibited growth phenotypes indicative of reduced m1A formation.In addition, recombinant mutant enzymes had reduced in vitro Mtase activity.We demonstrate that the mutations introduced do not prevent heteroligomer formation and do not disrupt binding of the cofactor S-adenosyl-L-methionine.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, Marquette University, P.O. Box 1881, Milwaukee, WI 53201, USA.

ABSTRACT
In Saccharomyces cerevisiae, a two-subunit methyltransferase (Mtase) encoded by the essential genes TRM6 and TRM61 is responsible for the formation of 1-methyladenosine, a modified nucleoside found at position 58 in tRNA that is critical for the stability of tRNA(Met)i The crystal structure of the homotetrameric m1A58 tRNA Mtase from Mycobacterium tuberculosis, TrmI, has been solved and was used as a template to build a model of the yeast m1A58 tRNA Mtase heterotetramer. We altered amino acids in TRM6 and TRM61 that were predicted to be important for the stability of the heteroligomer based on this model. Yeast strains expressing trm6 and trm61 mutants exhibited growth phenotypes indicative of reduced m1A formation. In addition, recombinant mutant enzymes had reduced in vitro Mtase activity. We demonstrate that the mutations introduced do not prevent heteroligomer formation and do not disrupt binding of the cofactor S-adenosyl-L-methionine. Instead, amino acid substitutions in either Trm6p or Trm61p destroy the ability of the yeast m1A58 tRNA Mtase to bind tRNA(Met)i, indicating that each subunit contributes to tRNA binding and suggesting a structural alteration of the substrate-binding pocket occurs when these mutations are present.

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trm6 mutants exhibit growth defects. (A) A trm6Δ strain over-expressing  and containing empty vector (Y351) or single copy TRM6 (Y353), trm6-416 (Y354), trm6-504 (Y360) or trm6-420 (Y367) was patched to a Sc-leu plate, grown, replica printed to a Sc-leu plate containing 5-FOA and incubated at 30°C for 3 days. (B)trm6-420 and trm6-504 strains that evicted the high-copy IMT4 plasmid (Y368 and Y361, respectively) were grown on YPD (Yeast extract/Peptone/Dextrose) at either 30 or 37°C for 3 days.
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Figure 2: trm6 mutants exhibit growth defects. (A) A trm6Δ strain over-expressing and containing empty vector (Y351) or single copy TRM6 (Y353), trm6-416 (Y354), trm6-504 (Y360) or trm6-420 (Y367) was patched to a Sc-leu plate, grown, replica printed to a Sc-leu plate containing 5-FOA and incubated at 30°C for 3 days. (B)trm6-420 and trm6-504 strains that evicted the high-copy IMT4 plasmid (Y368 and Y361, respectively) were grown on YPD (Yeast extract/Peptone/Dextrose) at either 30 or 37°C for 3 days.

Mentions: Although TRM6 is an essential gene, a trm6 deletion (trm6Δ) strain is viable when a high-copy plasmid containing IMT4, which encodes , is present (7). To demonstrate whether or not mutations in Trm6p affect m1A58 Mtase activity, a trm6Δ strain that over-expresses from a plasmid marked with URA3 (Y350) was transformed with a single copy LEU2 marked plasmid containing either TRM6 (Y353), trm6-416 (Y354), trm6-504 (Y360) or trm6-420 (Y367). Expression of Trm6p was found to be similar between the wild-type and mutant strains (data not shown). These strains were then evaluated on plates containing 5-fluoroorotic acid (5-FOA), which selects against URA3. Patches of cells grown on synthetic complete media lacking uracil and leucine (Sc-ura-leu) were replica printed to Sc-leu plates containing 5-FOA. Under these conditions, expression of TRM6 permitted growth throughout the patch, indicating the URA3 marked plasmid encoding had been readily evicted from these cells (Figure 2A). The strains expressing trm6-504 and trm6-420 were able to form papillae, but not a confluent patch, indicating only occasional eviction of the URA3 plasmid. The trm6-416 strain and the trm6Δ strain carrying an empty vector (Y351) were unable to grow (Figure 2A). The growth of cells containing TRM6 is expected because a functional m1A58 Mtase would be present and over-expression of would no longer be required. The limited growth of the trm6-504 and trm6-420 mutants suggests the m1A58 Mtase has reduced activity, while the complete lack of growth of the trm6-416 strain suggests the enzyme is no longer functional. The phenotypes observed using 5-FOA selection were the first indication that the mutations created in Trm6p had a detrimental effect on m1A58 Mtase activity.Figure 2.


Conserved amino acids in each subunit of the heteroligomeric tRNA m1A58 Mtase from Saccharomyces cerevisiae contribute to tRNA binding.

Ozanick SG, Bujnicki JM, Sem DS, Anderson JT - Nucleic Acids Res. (2007)

trm6 mutants exhibit growth defects. (A) A trm6Δ strain over-expressing  and containing empty vector (Y351) or single copy TRM6 (Y353), trm6-416 (Y354), trm6-504 (Y360) or trm6-420 (Y367) was patched to a Sc-leu plate, grown, replica printed to a Sc-leu plate containing 5-FOA and incubated at 30°C for 3 days. (B)trm6-420 and trm6-504 strains that evicted the high-copy IMT4 plasmid (Y368 and Y361, respectively) were grown on YPD (Yeast extract/Peptone/Dextrose) at either 30 or 37°C for 3 days.
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Related In: Results  -  Collection

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Figure 2: trm6 mutants exhibit growth defects. (A) A trm6Δ strain over-expressing and containing empty vector (Y351) or single copy TRM6 (Y353), trm6-416 (Y354), trm6-504 (Y360) or trm6-420 (Y367) was patched to a Sc-leu plate, grown, replica printed to a Sc-leu plate containing 5-FOA and incubated at 30°C for 3 days. (B)trm6-420 and trm6-504 strains that evicted the high-copy IMT4 plasmid (Y368 and Y361, respectively) were grown on YPD (Yeast extract/Peptone/Dextrose) at either 30 or 37°C for 3 days.
Mentions: Although TRM6 is an essential gene, a trm6 deletion (trm6Δ) strain is viable when a high-copy plasmid containing IMT4, which encodes , is present (7). To demonstrate whether or not mutations in Trm6p affect m1A58 Mtase activity, a trm6Δ strain that over-expresses from a plasmid marked with URA3 (Y350) was transformed with a single copy LEU2 marked plasmid containing either TRM6 (Y353), trm6-416 (Y354), trm6-504 (Y360) or trm6-420 (Y367). Expression of Trm6p was found to be similar between the wild-type and mutant strains (data not shown). These strains were then evaluated on plates containing 5-fluoroorotic acid (5-FOA), which selects against URA3. Patches of cells grown on synthetic complete media lacking uracil and leucine (Sc-ura-leu) were replica printed to Sc-leu plates containing 5-FOA. Under these conditions, expression of TRM6 permitted growth throughout the patch, indicating the URA3 marked plasmid encoding had been readily evicted from these cells (Figure 2A). The strains expressing trm6-504 and trm6-420 were able to form papillae, but not a confluent patch, indicating only occasional eviction of the URA3 plasmid. The trm6-416 strain and the trm6Δ strain carrying an empty vector (Y351) were unable to grow (Figure 2A). The growth of cells containing TRM6 is expected because a functional m1A58 Mtase would be present and over-expression of would no longer be required. The limited growth of the trm6-504 and trm6-420 mutants suggests the m1A58 Mtase has reduced activity, while the complete lack of growth of the trm6-416 strain suggests the enzyme is no longer functional. The phenotypes observed using 5-FOA selection were the first indication that the mutations created in Trm6p had a detrimental effect on m1A58 Mtase activity.Figure 2.

Bottom Line: Yeast strains expressing trm6 and trm61 mutants exhibited growth phenotypes indicative of reduced m1A formation.In addition, recombinant mutant enzymes had reduced in vitro Mtase activity.We demonstrate that the mutations introduced do not prevent heteroligomer formation and do not disrupt binding of the cofactor S-adenosyl-L-methionine.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, Marquette University, P.O. Box 1881, Milwaukee, WI 53201, USA.

ABSTRACT
In Saccharomyces cerevisiae, a two-subunit methyltransferase (Mtase) encoded by the essential genes TRM6 and TRM61 is responsible for the formation of 1-methyladenosine, a modified nucleoside found at position 58 in tRNA that is critical for the stability of tRNA(Met)i The crystal structure of the homotetrameric m1A58 tRNA Mtase from Mycobacterium tuberculosis, TrmI, has been solved and was used as a template to build a model of the yeast m1A58 tRNA Mtase heterotetramer. We altered amino acids in TRM6 and TRM61 that were predicted to be important for the stability of the heteroligomer based on this model. Yeast strains expressing trm6 and trm61 mutants exhibited growth phenotypes indicative of reduced m1A formation. In addition, recombinant mutant enzymes had reduced in vitro Mtase activity. We demonstrate that the mutations introduced do not prevent heteroligomer formation and do not disrupt binding of the cofactor S-adenosyl-L-methionine. Instead, amino acid substitutions in either Trm6p or Trm61p destroy the ability of the yeast m1A58 tRNA Mtase to bind tRNA(Met)i, indicating that each subunit contributes to tRNA binding and suggesting a structural alteration of the substrate-binding pocket occurs when these mutations are present.

Show MeSH
Related in: MedlinePlus